Preparation Of Corn Peptides With High ACE Inhibiting Activity By Membrane Separation And Enzyme Treatment

In order to utilize the plant protein, the corn proteins were extracted from byproducts producing corn starch. The high-value bioactive corn peptides (CP)---angiotensin converting enzyme (ACE) inhibiting peptides were prepared by enzymolysis. First the five different proteases were screened. Second, the ACE inhibitory activity of CP was improved by orthogonal test and membrane separation. Third, the L-proline were connected to the peptide end by plastein reaction, this method could further enhance the ACE inhibitory activities of CP. The last, the amino acid sequences of peptides after ultrafiltration and plastein were analyzed by HPLC-MS/MS, the structure-effect of corn peptides were discussed. The main results were shown as follows:1. Study on the hydrolysis conditions:The degree of hydrolysis (DH) and the ACE inhibition activities of CP were compared after hydrolysis with Nutrase, Alcalase, Flavourzyme, Pepsin and Trypsin. The Alcalase was selected be using in this experiment. Then, to obtain the CP with high ACE inhibitory activity, the optimum hydrolysis conditions were:ratio of enzyme and substrate:1.00%, ratio of materiel and water:1:25, temperature:55℃, pH 8.0, time:4h. Under this condition, ACE inhibition rate of CP was 67.01%.The hydrolyzates were separated by Sephadex G-15 gel chromatography, the molecular weight range of CP with higher ACE inhibitory activity components were 5647-1005Da.2. Study on the ultrafiltration conditions:the corn protein hydrolyzates were fractioned by three kinds of ultrafiltration membranes (molecular weight cut off, MWCO 5kDa,3kDa, 1kDa), and the three permeate products (Mw<5 kDa,<3 kDa,<1 kDa) were obtained. The peptides content and ACE inhibitory activities were compared before and after fractionation, and filtrate of 3kDa membrane had higher ACE inhibitory activity. Then it was done that the digestion tests in vitro of the hydrolyzates before and after fractionation with pepsin, trypsin and pepsin & trypsin, and filtrate of 3kDa membrane had lower activity loss (11.98%). The ultrafiltration membrane with MWCO 3kDa was the most suitable working membrane. The CP solutions were treated by ultrafiltration membrane with MWCO 3kDa. And the optimum parameters for ultrafiltration were: concentration ratio 3:1, pH 5.5, temperature 35℃, under which the ACE inhibitory rate of CP fraction was 82.27%.3. Study on Plastein reaction:Plastein reaction is a reverse reaction of protein hydrolysis. L-proline was added in order to further enhance the ACE inhibitory activity of CP. The ACE inhibitory activities of CP increased after modified, because the proline were integrated into the C-terminal or N-terminal of peptides. The optimal reaction conditions of plastein were as follow:corn peptides concentration 25%, time 6h, proline concentration 2.5%, and the ACE inhibitory rate was 92.10% under these conditions. The activity enhanced 27.24% comparing to original corn protein hydrolyzates.4. Study on desalination by nanofiltration membrane:The desalination and peptides retention rate were compared under three types of operation model (dilution-concentration-dilution batch volume-constant diafiltration, concentration-dilution-concentration batch volume-constant diafiltration, continuous volume-constant diafiltration). The continuous diafiltration was the best process to desalinate, after 95min, the peptides retention rate was 98.49%, desalination rate was 67.64%. Then, the desalination effects of hydrolyzates were compared under different pH using the best process. Under pH 7, the after 80min, desalination rate could reach 87.80%.5. Study on the Structure of ACE inhibitory corn peptides:First, the amino acid analysis results of CP by different treatment (Original hydrolysates, after Mw<3 kDa ultrafiltration and Plastein treatment) showed that hydrophobic amino acids, aromatic amino acid, proline, peptide purity and ACE inhibition rate all progressively increased. Then, the two kinds of corn peptides’structures (after Mw<3 kDa ultrafiltration and Plastein treatment) were analyzed by HPLC-MS/MS, and their amino acid sequences were Gln-Leu-Leu-Pro-Phe and Pro-Pro-Ala-Leu-Gly-Phe.